Many proteins essential for spliceosome assembly and splicing have been identified, and numerous human homologs of essential yeast splicing factors are now known (for reviews, see references 19
, and 29
). Among the best characterized of these are the components of U2 snRNP (for reviews, see references 19
, and 29
). In mammals, functional 17S U2 snRNP can be assembled from 12S U2 snRNP and two essential splicing factors, SF3a and SF3b (5
). SF3a has been purified to homogeneity and contains three proteins (SF3a60, SF3a66 and SF3a120) (5
). SF3b has been purified through multiple chromatographic steps but has not been purified to homogeneity (5
). The components thought to constitute SF3b were identified by comparing purified 17S U2 snRNP and the spliceosomal complex A (for reviews see references 14
). The abundant proteins common to both of these complexes are referred to as SF3b 53, 120, 150, and 160 in 17S U2 snRNP and SAPs 49, 130, 145 and 155, respectively, in the spliceosome (we use the latter nomenclature here) (2
). Further evidence that at least two of these proteins are components of SF3b came from the observation that SAPs 49 and 145 interact directly with each other (7
). In addition, SAPs 49, 145, and 155, as well as all three SF3a subunits, can be UV cross-linked to the region surrounding the branch site in the spliceosomal complex A (11
). Thus, these proteins are all located next to one another in functional spliceosomal complexes, consistent with the notion that they are present in a complex. Despite all of the circumstantial evidence that SAPs 49, 130, 145, and 155 correspond to SF3b, it remains to be established whether any or all of these proteins are indeed components of a single protein complex.
All of the mammalian SF3a components and three of the putative SF3b components (SAPs 49, 145, and 155) have been cloned (19
). In addition, yeast counterparts of SF3a have been identified and shown to be essential for viability (for a review, see reference 19
). In contrast to SF3a, none of the putative SF3b components were identified in the early genetic screens for yeast splicing factors. However, the likely Saccharomyces cerevisiae
homologs of SAPs 145 and 155, scSAP 145 and scSAP 155, were identified in the GenBank database on the basis of their similarity to the corresponding mammalian proteins (7
). One of these proteins, scSAP 145, was subsequently found to be the same as CUS1, a protein identified as a suppressor of a U2 snRNA mutation (27
). scSAP 145 is essential for A complex assembly in yeast (27
). scSAP 49/HSH49 was also identified in the database and shown to be essential for viability in yeast (15
). Yeast SAPs 49 and 145, like their mammalian counterparts, interact directly with each other via protein-protein interactions and thus are presumed to be components of a yeast SF3b complex (10
). It is not yet known whether scSAP 155 is essential in yeast or whether a yeast counterpart of SAP 130 exists.
Here we report the isolation of a cDNA encoding SAP 130. Using antibodies to this protein, as well as antibodies to other putative SF3b components, we showed that SAPs 130, 145, and 155 are present in a protein complex and that SAPs 130 and 155 interact (directly or indirectly) with each other within this complex. Together with previous work, our data provide strong evidence that SAPs 49, 130, 145, and 155 are components of SF3b. We have also completed the description of the yeast SF3b counterparts by identifying the S. cerevisiae homolog of SAP 130 and showing that it and scSAP 155 are essential in yeast. Thus, together the data indicate that the spliceosomal proteins SAPs 49, 130, 145, and 155 are components of a highly conserved protein complex essential for splicing.